In scanning microscopy, an object to be examined is illuminated with a light beam in order to observe the reflection light or fluorescent light emitted by the sample. The focus of the illumination light beam is moved in an object plane with the help of a controllable beam deflector, generally by tipping two mirrors, whereby the axes of deflection are mostly positioned perpendicular to each other so that one mirror deflects in the x-direction and the other in the y-direction. The mirrors can, for example, be tipped with the help of galvanometric positioners. Measurement of the power of the light coming from the object is dependent on the scanning position of the illumination light beam.
In confocal scanning microscopy in particular, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optic with which the light from the source is focused on a pinhole aperture—the so-called excitation aperture—, a primary beam splitter, a beam deflector to control the beam, a detection aperture, and at least one detector to detect detection light or fluorescent light that is emitted by the object. The primary beam splitter, via which the illumination light beam is coupled, is implemented such that the illumination light is largely reflected, whereas the reflection light and/or fluorescent light largely passes the primary beam splitter.
Category-defining devices that offer the possibility of object manipulation are used particularly in cell biology, for example, to manipulate the insides of unopened cells. Here, two different methods of manipulation, in particular, are usual. On the one hand, objects or object regions are illuminated with focused infrared light, as a result of which individual particles of the objects or object region, respectively, are captured in the proximity of the manipulation focus, and can then be moved in unison with the focus when the position of the manipulation focus is changed in the focal plane (optical tweezers), so that they may, for example, be impinged upon by a force. If an object region is impinged upon by a pulsed focused UV light, biological material can be cut or perforated as a result of the high energy density of UV light (nanoscalpel).
A multiplicity of other applications is known in scanning microscopy in which a manipulation light beam is used in addition to the illumination light beam for manipulating objects.
DE 100 39 520 A1 describes a device for examining and manipulating microscopic objects, in which the manipulation light beam and the illumination light beam are merged by means of a scanning mirror of a beam deflector. Here, the scanning mirror is designed to be transparent to the light from the manipulation light source, while reflecting the light from the illumination light source. The known device is disadvantageous to the extent that a total of two beam deflectors are needed, whereby one beam deflector controls the manipulation light beam, and the other beam deflector the illumination light beam. The need for two beam deflectors makes the known device both expensive to produce and time-consuming to operate because both beam deflectors must be precisely synchronized in order to keep the beam angle between the manipulation light beam and the illumination light beam constant during the entire scanning process.
A scanning microscope with a manipulation light beam and an illumination light beam is also known from DE 102 33 549 A1, whereby the merging of both light beams is done with the help of the primary beam splitter. The primary beam splitter serves primarily to separate the illumination light beam and the detection light beam. For this purpose, the primary beam splitter is generally implemented either as a band-pass filter or cut-off filter or as a partially transmitting neutral splitter that reflects the light of the illumination light beam and permits the light of the detection light beam to pass. Because the spectral edges of the beam splitter are not infinitely steep, but rather exhibit a certain slope, the wavelengths or wavelength ranges, respectively, of the illumination light, the detection light, and the manipulation light can, under certain circumstances when the manipulation light beam is also coupled, be selected only from a narrowly delimited range, to be precisely coordinated with the spectral characteristics of the primary beam splitter.